Journal of Plant Physiology 177 (2015) 30–43

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Photosynthesis-dependent physiological and genetic crosstalk between cold acclimation and cold-induced resistance to fungal pathogens in triticale (Triticosecale Wittm.) a,b,∗ a c ´ ˛ ˛ Magdalena Szechynska-Hebda , Iwona Wasek , Gabriela Gołebiowska , Ewa Dubas a , a a,c ˙ ˛ Iwona Zur , Maria Wedzony a

Institute of Plant Physiology, Polish Academy of Sciences, Niezapominajek 21, 30-239 Krakow, Poland Department of Plant Genetics, Breeding and Biotechnology, Faculty of Horiculture Biotechnology and Landscape Architecture, Warsaw University of Life Sciences, Nowoursynowska 159, 02-776 Warszawa, Poland c ˙ Pedagogical University of Krakow, Podchora˛ zych 2, 30-084 Krakow, Poland b

a r t i c l e

i n f o

Article history: Received 21 June 2014 Received in revised form 26 November 2014 Accepted 6 December 2014 Available online 14 January 2015 Keywords: Cold acclimation Cold-induced resistance Quantitative trait loci Peroxidases Photosynthesis

a b s t r a c t The breeding for resistance against fungal pathogens in winter triticale (Triticosecale Wittm.) continues to be hindered by a complexity of the resistance mechanisms, strong interaction with environmental conditions, and dependence on the plant genotype. We showed, that temperature below 4 ◦ C induced the plant genotype-dependent resistance against the fungal pathogen Microdochium nivale. The mechanism involved, at least, the adjustment of the reactions in the PSII proximity and photoprotection, followed by an improvement of the growth and development. The genotypes capable to develop the cold-induced resistance, showed a higher maximum quantum yield of PSII and a more efficient integration of the primary photochemistry of light reactions with the dark reactions. Moreover, induction of the photoprotective mechanism, involving at least the peroxidases scavenging hydrogen peroxide, was observed for such genotypes. Adjustment of the photosynthesis and stress acclimation has enabled fast plant growth and avoidance of the developmental stages sensitive to fungal infection. The same mechanisms allowed the quick regrow of plants during the post-disease period. In contrast, genotypes that were unable to develop resistance despite cold hardening had less flexible balancing of the photoprotection and photoinhibition processes. Traits related to: photosynthesis-dependent cold-acclimation and cold-induced resistance; biomass accumulation and growth; as well as protection system involving peroxidases; were integrated also at a genetic level. Analysing 95 lines of the mapping population SaKa3006 × Modus we determined region on chromosomes 5B and 7R shared within all tested traits. Moreover, similar expression pattern of a set of the genes related to PSII was determined with the metaanalysis of the multiple microarray experiments. Comparable results for peroxidases, involving APXs and GPXs and followed by PRXs, indicated a similar function during cold acclimation and defense responses. These data provide a new insight into the cross talk between cold acclimation and cold-induced resistance in triticale, indicating a key role of photosynthesis-related processes. © 2015 Elsevier GmbH. All rights reserved.

Introduction Snow mold caused by Microdochium nivale (Fr.) Samuels and Hallett is the most widespread seedling disease of winter cereals.

∗ Corresponding author at: Institute of Plant Physiology Polish Academy of Sciences Niezapominajek 21 30-239 Krakow, Poland. Tel.: +48 12 425 33 01; fax: +48 12 425 18 44. E-mail addresses: [email protected], [email protected] ´ (M. Szechynska-Hebda). 0176-1617/© 2015 Elsevier GmbH. All rights reserved.

M. nivale is a fungal psychrophilic pathogen that is able to invade under the snow or during rainy, winter weather. The conditioning of the plant seedlings in low but positive temperature in the presence of light promotes genotype-dependent resistance to M. ˛ ˛ ´ nivale infection (Gołebiowska and Wedzony, 2009; SzechynskaHebda et al., 2011). For cultivars able to develop resistance after cold treatment, increasing the duration of hardening from 0 to 98 days enhances plant survival and green biomass production almost linearly. In contrast, a period of 98 days still does not enable the susceptible cultivar to survive infection and regrowth of ´ seedlings (Szechynska-Hebda et al., 2013). It has been shown that

M. Szechy´ nska-Hebda et al. / Journal of Plant Physiology 177 (2015) 30–43

M. nivale behaves as a biotroph in plants that are able to develop cold-induced resistance and as a necrotroph in tissues that are sus´ ceptible to infection (Szechynska-Hebda et al., 2013). This fungal behavior has been linked to passive defense developed under light and cold temperatures in host leaves, and particularly to physical and chemical properties of the leaf surface as well as composition ´ and structure of cell wall in the leaf interior (Szechynska-Hebda et al., 2013). The cold-induced resistance mechanism is dependent on the upregulation of a wide range of stress-response genes (Gaudet et al., 2011). Recent research has identified genes encoding regulators of cold hardening and cold acclimation, e.g. COR (cold-responsive), LTI (low temperature-induced), RAB (responsive to abscisic acid), KIN (cold-induced) or ERD (early responsive to dehydration) proteins, antifreeze proteins, soluble carbohydrates contributing to an increase in cell osmotic potential, protein chaperones, and RNA chaperones (reviewed in detail by Ruelland et al., 2009). Moreover, it has been shown that cold acclimation of winter crops involves genotype-dependent changes in hormone composition, stress-related protective substances (proline, phenolics), and cellular redox status (Majláth et al., 2012). Although a large number of genetic and biochemical responses triggered by cold hardening can be universal and useful for a plant in its defense processes, the exact mechanisms involved in cold-induced resistance to pathogens still remains to be identified. Some evidence indicates the key role of photosynthesis in the induction of plant resistance by cold. First, it is known that the acquisition of cold acclimation and resistance requires light (Ruelland et al., 2009), which is a driving force for photosynthesis. The hardening of cereals, such as rye or wheat, is much more effective under normal light conditions than under low light conditions (Janda et al., 2014). Light has a significant, genotypedependent effect on the expression level of genes involved in the hardening process; but also genes that reflect overlapping between the signaling routes of abiotic stress tolerance and pathogen defense (Majláth et al., 2012). Among others, the regulation of photosynthesis-related genes, stress-related genes, and genes related to the N-metabolism was recognized as a necessary condition of the acclimation responses (Rapacz et al., 2008; Liu et al., 1998; Janda et al., 2014) and defense responses (Bilgin et al., 2010). Second, the excitation level of photosystems is a critical factor in both, abiotic stress acclimation and resistance induction ´ ´ et al., 2010; Karpinski and (Huner et al., 1993; Szechynska-Hebda ´ Szechynska-Hebda, 2010). Light energy trapping by the antenna of PSII and PSI, use of this energy to drive charge separation within the reaction center cores, are both largely temperature-independent, but in contrast, low temperatures inhibit thylakoid electron transport by increasing membrane viscosity and restricting the diffusion of plastoquinone. Therefore, under cold conditions, an imbalance occurs within the amount of light energy absorbed by primary photochemical reactions in PSII and PSI, the transformation of this energy into NADP and ATP, and its utilization in metabolism (Ruelland et al., 2009). The excess excitation energy and reduction state of PS II has been shown to correlate with the induction ´ of certain light-, cold- and defense-related genes (Szechynska´ ´ Hebda et al., 2010; Karpinski and Szechynska-Hebda, 2010; Janda et al., 2014). Third, photosynthesis-originated ROS and mechanisms of their scavenging determine the outcome of cold stress and pathogen-generated injuries. Excess excitation energy induced by the cold under light conditions in the light-harvesting chlorophyll antennae can favor the production of ROS, inactivation of PSII, chloroplast membrane damages and photoinhibition. On a time scale of minutes, plants can acclimate by diverting energy from PSII to PSI through state transitions or by dissipating excess energy as heat by non-photochemical quenching. On a longer time scale, photosynthetic acclimation may occur as a consequence of a reduction


in PSII antenna size and an increase in the capacity for ROS scavenging (Huner et al., 1993; Ruelland et al., 2009). In particular, the expression of the genes encoding iron superoxide dismutase, glutathione-dependent peroxidases and ascorbate peroxidases was specifically regulated during the acquired acclimation to abiotic stress and acquired resistance to pathogens (Ruelland et al., 2009; ´ ´ and Karpinski, 2013). Fourth, cold induces Szechynska-Hebda changes in the plant carbohydrate metabolism, an outcome of photosynthesis (Ruelland et al., 2009; Janda et al., 2014). Genotypedependent acclimation involves modification of both protective (cytosolic) and structural (cell wall) carbohydrates, thus influences the level of plant’s resistance or susceptibility (Majláth et al., 2012; ´ Szechynska-Hebda et al., 2013). Fifth, recent results showed that besides changes in PSII and alterations in (anti)oxidative status, several other mechanisms originated in chloroplasts, including salicylic acid metabolism may also contribute to the resistance ´ induced by cold and/or light (Karpinski et al., 2013; Janda et al., 2014). Light is known to affect SA metabolism during cold hardening and it has also been shown that the overexpression of a salicylic acid (SA)-inducible Dof (DNA binding with one finger) transcription factor (OBP3) resulted in growth defects. The signaling sugar molecules are yet another potential regulator of Dof domain transcription factors (Kang and Singh, 2000; Janda et al., 2014). Thus, it can be assumed that role of SA and signaling sugar can be physiologically integrated and play a role in the control of nuclear genes and growth regulation induced by light and low temperatures. Since a plant must adjust its photosynthetic activity during hardening in order to achieve appropriate acclimation and defense responses (Huner et al., 1993), in the present studies we tried to answer the following questions: (1) What photosynthesis-related mechanisms are common and required for both, plant acclimation to cold and resistance to fungal pathogen? (2) Can plants adjust their photosynthesis to enable fast plant growth and to avoid developmental stages that are sensitive to fungal infection? (3) How do changes in photosynthesis during cold hardening process contribute to resistance reactions and plant regrowth after infection? and (4) Is the peroxidases activity a part of the strategies that help to reduce oxidative stress during cold hardening and cold-induced resistance? With a set of 95 doubled haploid (DH) lines originating from a cross between ×Triticosecale Wittm. SaKa3006 and Modus, a comparable analysis of traits between hardened and unhardened plants was performed. As a result, we determined a tight correlation between the cold acclimation and cold-induced resistance on both the physiological level (growth and development, photosynthesis efficiency expressed as Fv /Fm and PI, peroxidases activity) and the genetic level (QTL analysis of physiological traits and metaanalysis of transcripts related to PSII and peroxidases).

Materials and methods Plant material and growth conditions A population of 95 DH lines originated from an intercultivar cross between two unrelated hexaploid winter triticales (Triticosecale Wittm.) was studied (Tyrka et al., 2011). The breeding line SaKa3006 (SaKa Pflanzenzucht GbR, Germany) was used as a female parent and the cultivar Modus (registered in Poland, released by Plant Breeding Strzelce Ltd.) was the pollen parent. The cultivars SaKa3006 and Modus were chosen as parents because of their different origin and different responses to cold-induced resistance to Microdochium nivale. Modus has been described as a cultivar able to develop partial resistance after cold treatment, and SaKa3006 as a cultivar susceptible to fungal infection despite plant hardening with cold. The seeds of the parent plants and lines of


M. Szechy´ nska-Hebda et al. / Journal of Plant Physiology 177 (2015) 30–43

the SaKa3006 × Modus population were surface-sterilized in 0.05% (v/v) NaOCl for 15 min, rinsed with sterile distilled water and germinated on moistened filter paper. After two days the seedlings were planted in a mixture of soil:peat:sand (2:2:1 v/v/v) at pH 5.8. The plants grew in an isolated, fully controlled climatic chamber for 2 weeks under light, 100 ± 10 ␮mol (quantum) m−2 s−1 PAR, 8 h/16 h (day/night), at 20 ◦ C/17 ◦ C and a relative humidity of RH = 60–67%. Then, they were subjected to a pre-hardening period (12 ◦ C for 7 days) and hardening (4 ◦ C for 14 and 28 days) in the same light regime (Supplementary Fig. 1A and B). Unhardened plants grew 4 weeks under light, 100 ± 10 ␮mol (quantum) m−2 s−1 PAR, 8 h/16 h (day/night), at 20 ◦ C/17 ◦ C to achieve the same physiological age as hardened plants (similar leaves number) (Supplementary Fig. 1C). Plant inoculation with pathogen and regrowth Mycelium was derived from the monosporal isolate of M. nivale with a high virulence (monosporal isolate no. 38z/5a/01). The isolate was collected from rye by Professor Maria Pronczuk from the Institute of Plant Breeding and Acclimatization, Radzikow, Poland. The fungus was grown for 10 days in darkness at 20 ◦ C on potato dextrose agar (PDA) medium, and then 14 days in soil mixed with wheat bran (10:1). Hardened and unhardened seedlings (Supplementary Fig. 1A and C, respectively) were inoculated by spreading 1 g of soil/wheat bran with mycelium on the soil surrounding the plant. Each pot was then covered with moistened blotting paper and plastic foil to imitate conditions occurring under a snow cover and kept at 4 ◦ C in darkness. Another set of hardened plants were treated the same way, except for the infection (Supplementary Fig. 1 B). The covers were removed after 21 days of incubation, the seedlings were cut 4 cm above ground level and allowed to regrow for 14 days under optimal conditions for recovery: 100 ± 10 ␮mol (quantum) m−2 s−1 PAR, 8 h/16 h (day/night), at 20 ◦ C/17 ◦ C and a relative humidity of RH = 60–67%. The number of plants with regenerated shoots was used as an indicator of resistance. Morphological and developmental traits Seeds were harvested from plants grown under controlled greenhouse conditions and preserved dry for 3 months. A thousand seed weight (TSW) was determined before sowing. Rate of the seedling germination was characterized by the time (h) needed to reach 50% of final germination (T50) and the percentage of germinated seeds (total germination). Shoot and leaf formation was presented as: an average of the daily increase in shoot length (cm/24 h) recorded during a period of 10 consecutive days (growth rate), the number of leaves and shoots (leaves no and shoots no, respectively) calculated for 4-week-old plants, fresh weight and dry weight (drying: 24 h, 105 ◦ C) of 4-week-old seedlings. Root formation, expressed by root length, was measured for 9-week old plants. Chlorophyll a fluorescence parameters Measurements were performed on the middle section of the second fully expanded leaf of the unhardened seedlings (20 ◦ C/17 ◦ C; day/night), pre-hardened seedlings (12 ◦ C) and hardened seedlings (4 ◦ C) for 14 and 28 days. The chlorophyll a fluorescence was measured with a Handy PEA fluorometer (Hansatech Ltd. Kings Lynn, UK) (Strasser et al., 2004). Before measurements the LED light source of the fluorometer was calibrated using an SQS light meter (Hansatech Ltd. Kings Lynn). All measurements were taken on dark-adapted leaves (30 min in the leaf clip) using a saturating PPFD of 800 ␮mol m−2 s−1 , and the induction time was 1 s. The maximum quantum yield of PSII (Fv /Fm = (Fm − F0 )/Fm ) and the performance index (potential) for energy conservation from photons

absorbed by PSII to the reduction of intersystem electron acceptors (PI = ((1 − (F0 /Fm ))/M0 /VJ ) × (Fm − F0 )/F0 × (1 − VJ )/VJ ) were measured. Fm represents maximal fluorescence intensity when all PSII RCs are closed, F0 —minimal fluorescence when all PSII RCs are open, M0 —initial slope of the fluorescence transient normalized on the maximal variable fluorescence Fv , VJ —relative variable fluorescence at the J-step. All fluorescence measurements were taken in 20 replications from different plants and different plant sets (Supplementary Fig. 1A and B). Peroxidase analysis The measurements of peroxidase activity were performed for the parental genotypes (SaKa3006 and Modus) and for 30 lines of the DH population. A total of 30 lines were chosen from each of the following groups: 10 lines susceptible to infection despite hardening including SaKa3006, 10 lines moderate in their response to infection including Modus, and 10 lines capable to acquire cold-induced resistance against infection. The other experimental conditions as well as experiment design were identical to those described above. Measurements of total peroxidase activity were based on the modified method of Lück (1962). Samples were homogenized at 4 ◦ C with 0.05 M phosphate buffer (PB, pH 7.0) containing 0.1 mM EDTA and 1% PVP-40. The proportion of plant biomass to extraction buffer (w:v) was 1:2. The homogenate was centrifuged for 10 min at 15,000 × g and the supernatant was dialyzed overnight in PB. The reaction mixture contained 1% pphenylenediamine (8 ␮l), PB (2 ml) and supernatant (8 ␮l). The reaction was started in the presence of 0.1 mM H2 O2 , and then measured during 2 min at 460 nm using a Perkin Elmer UV–vis spectrophotometer. Protein determination was assayed by Bradford’s dye-binding technique (Bradford, 1976) with bovine serum albumin as a protein standard. Construction of the genetic map and QTL identification The studies exploited the first high-density triticale linkage map that was constructed for 95 DH lines derived from the SaKa3006 and Modus F1 hybrid (Tyrka et al., 2011). On multiple-mapping approaches, a total of 1568 markers were ordered in 21 linkage groups using a logarithm of odds (LOD) > 3.5 and assigned to the A, B, and R genomes including 155 simple sequence repeat (SSR), 1385 diversity array technology (DArT) and 28 amplified fragment length polymorphism (AFLP) markers. The sequence of markers was recalculated and the distances between loci were determined with the Kosambi function. The chromosomal locations and order of the markers in those studies were in accordance with Tyrka et al. (2011). The map covers 2397 cM and the average distance between markers is 4.1 cM. This map was used for present study, which aimed to identify regions in the triticale genome that influence traits of physiological importance related to acclimation and defense responses. The QTLs were calculated for the following traits related to photosynthesis efficiency: chlorophyll a fluorescence parameters Fv /Fm and PI in plants before hardening, pre-hardened for 7 days and hardened for 14 and 28 days. Moreover, QTLs for peroxidase activity were studied. Several parameters associated with biomass accumulation and M. nivale infection were also analyzed to determine QTLs: fresh weight and dry weight; leaves formation; and the number of plants with regenerated shoots (resistance), all of them measured on the 14th day of regrowth after infection. Statistical analysis The experiments were performed in a randomized complete block design with eighteen replicates (three rows with six seedlings

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each) for particular lines and parents. Two independent experiments were conducted during autumn and spring in isolated growth chambers. The correlation coefficients calculated between the seasons for particular traits ranged from 0.5 to 0.99. Standard deviation, the correlation coefficients (R2 ) between particular traits, and Student’s t-test were calculated using Microsoft Excel 2010. The data set was subjected to analysis of variance (ANOVA) to determine specific effects of the genotype (i.e. the DH line) (Statistic 8.0). For each parameter, the normal distribution of scores was verified by the Shapiro–Wilk test in order to validate the use of the parametric tests. The effect of the tested variable/variables was examined by one-way or multi-factor analysis of variance (ANOVA). QTLs were identified using Windows QTLCartographer version 2.5 (Wang et al., 2007) and the results were analyzed using singlemarker analysis (SMA) and composite interval mapping (CIM). SMA analysis fits the data to a simple linear regression model. The CIM method, in turn, determines the linkage between QTL and the markers limiting the designated interval on the chromosome map. The threshold logarithm of the odds (LOD) scores was calculated based on 1000 permutations and 1 cM walk speed. A QTL was accepted when the LOD score was higher than 2.5. Percentage of the phenotypic variation was calculated with a single-factor regression (R2 ). Meta-analysis of peroxidase genes Meta-analytical tool of the Genevestigator (https://www. was used to study the selected transcriptomes of winter wheat (Triticum aestivum L.). The average expression of genes was calculated for total number of transcripts related to peroxidases, and then for transcripts related to ascorbic peroxidase (APX), glutathione peroxidase (GPX) and peroxiredoxins (PRX). Similarly, transcripts related to PSII were chosen for microarray metaanalysis. Meta-analysis of microarray experiments was performed by choosing the microarray experiments for wild type T. aestivum plants (platform TA 55 K: Wheat Genome Array) that provide appropriate stress conditions and 120 perturbations with the total sample number 490 were selected. 104 of them were chosen from the experiments focusing on the plant infection with fungal pathogens: Blumeria graminis, Fusarium graminearum, Puccinia striiformis, Puccinia triticina, Tilletia caries, according to experiments with accession numbers: TA-00003, TA-00005, TA-00009, TA-00010, TA-00013, TA-00014, TA-00022, TA-00023, TA-00024, TA-00027, TA-00031, TA-00032, TA-00036, TA-00044, TA-00046. Sixteen of them were chosen from the experiments concerning cold acclimation that were described with accession numbers: TA-00033 and TA-00043. All data were obtained from GV database as an average expression for samples treated with appropriate stress vs samples from control conditions. Results Correlation of growth-related traits with photosynthetic efficiency and resistance Table 1 summarizes the traits related to seed ability, germination and growth, the photosynthetic efficiency of seedlings expressed as chlorophyll a fluorescence parameters as well as the seedlings’ resistance to infection expressed as a percentage of regenerated plants. The studied traits were calculated as an average from two different seasons and showed a good fit to the normal distribution. All average values for the cultivar Modus, with exception of the time required to reach 50% germination, were higher in comparison with cultivar SaKa3006. The most important differences between the SaKa3006 and Modus traits were found in resistance


(91%), dry weight of 4-week-old seedlings (33.3%), the daily shoot growth rate (32%), and time required to reach 50% germination, T50 (27.7%). Other traits related to formation of the shoots, leaves and root were higher for Modus by about 14.6–21.6%. A traits analysis within the 95 DH lines showed great variation in all of the studied phenotypic parameters. Some of the lines had much lower average values than the parents and some of them exceeded the Modus traits. Two studied parameters of chlorophyll a fluorescence showed genotype-dependent responsiveness. Higher values of the maximum quantum yield of PSII (Fv /Fm ) were measured for Modus in comparison to SaKa3006. However, the differences were not as considerable as for the performance index (PI), an indicator of plant vitality. The DH progeny population presented a wide spectrum of responses: the mean Fv /Fm ranged from 0.815 to 0.826, and PI from 1.55 to 2.15 within particular lines. Cold-induced resistance to the fungal pathogen M. nivale was evaluated based on seedlings hardening (for 28 days), 3-week incubation with mycelium, and the regrowth ability after the controlled infection with the fungus. SaKa3006 and Modus differed in their regeneration potential after infection (Table 1 and Supplementary Fig. 2). The SaKa3006 plants were very susceptible and exhibited only 36.67% of survival, whereas 70% of the Modus plants regenerated in optimal conditions. Transgressive segregation was observed in the evaluated population, evident by results for individual DH lines. 3% of DH lines had lower survival capability than susceptible SaKa3006, and 77% of the lines had elevated regeneration abilities in comparison to the partly resistant Modus. 20% of lines express full resistance in the experimental conditions (data not presented in Table 1). Pearson’s correlation coefficients within the parameters of chlorophyll a fluorescence (Fv /Fm and PI), the resistance to the pathogen infection and traits related to the seedling growth were calculated for 95 DH lines (Table 1). Significant correlations (P < 0.05) were found amongst all of the traits. Plant germination (expressed as T50), growth rate and number of leaves per seedling as well as seedling dry weight were correlated relatively strongly with the chlorophyll a fluorescence parameters (0.499–0.678). Similarly, high values of the R coefficients were found, when these phenotypic traits were correlated with plant resistance to M. nivale (0.541–0.648). For both chlorophyll a fluorescence parameters and seedling resistance, the relationship with the number of seedling shoots and seedling fresh weight was moderate (0.325–0.463), while correlation to TSW and the length of seedling roots was the lowest (0.162–0.398). The percentage of regenerated plants after infection was also positively correlated with Fv /Fm and PI at a level of 0.4728 and 0.4897, respectively. Cross-sections of 3-day-old hypocotyls were performed for the lines chosen from each of the following groups: lines susceptible to infection despite hardening including SaKa3006, lines moderate in their response to infection including Modus, and lines capable to acquire cold-induced resistance against infection. Fig. 1 demonstrates fast chloroplasts development in coleoptile near vascular bundle sheath for moderate genotypes (Fig. 1G–H and J–K) and genotypes capable to acquire cold-induced resistance (Fig. 1M–N and P–Q). Similarly, the leaves development, their physiological and metabolic maturity progressed much more quickly for such genotypes (Fig. 1I, L, O and R) in comparison to genotypes susceptible to infection (Fig. 1C and F). Cold-induced resistance is related to photosynthesis efficiency Our earlier study showed that several mechanisms related to the basal resistance of winter triticale to M. nivale were cultivar-dependent and developed only during plant hardening ´ et al., 2013). Here, we linked the cold-induced (Szechynska-Hebda


M. Szechy´ nska-Hebda et al. / Journal of Plant Physiology 177 (2015) 30–43

Fig. 1. Representative examples of 3-days-old coleoptile demonstrating the chloroplasts development near vascular bundle sheath and the first leaves development. The cross-sections were performed for parental seedlings Saka3006 (A–C) and Modus (G–I) as well as in the DH line that is sensitive to infection despite hardening (D–F), line moderate in the resistance responses (J–L), and two lines able to acquire cold-induced resistance against infection (M–R). Chloroplasts (chlorophyll) are visualized by red autofluorescence under UV light and green color in bright field photos. Bars for left and middle column, 50 ␮m; bar for right column, 200 ␮m.

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Table 1 Statistical parameters for several morphological, developmental and physiological traits of parents (SaKa 3006, Modus) and 95 DH lines derived from two different seasons. All plants grew under standard laboratory conditions (100 ± 10 ␮mol m−2 s−1 PAR, 8 h/16 h day/night, at 20 ◦ C/17 ◦ C and a relative humidity of RH = 60–67%). The resistance was determined for plants treated as follow: hardened 28 days, infected with M. nivale, regown in optimal conditions for 14 days. The correlation coefficients (R2 ) between those traits and parameters of chlorophyll a fluorescence (Fv /Fm and PI) or seedlings resistance to infection were calculated for DH lines. Significance levels between parents were calculated according to t-Student test: * P ≤ 0.05; ** P ≤ 0.01, *** P ≤ 0.005. Traits

DH population

Parents Saka3006


Seeds ability 51.31 55.85 TSW Germination 65.50 51.29** T50 85.18 96.29* Total Shoots and leaves formation Growth rate 1.50 1.98* 3.14 3.72* Leaves no 2.05 2.46 Shoots no 0.96 1.10 FW 0.12 0.16** DW Roots formation 21.48 26.12 Length Photosynthesis efficiency 0.828 0.830 Fv /Fm 1.68 2.05** PI Seedlings regeneration after infection with M. nivale 36.67 70.00*** Resistance





R2 Fv /Fm










34.41 97.30

20.44 80.55

64.50 99.33

9.04 4.20

−0.542 0.438

−0.582 0.441

−0.648 0.626

2.31 3.77 2.66 1.15 0.16

1.50 2.94 1.97 0.77 0.12

3.16 4.65 3.38 1.97 0.24

0.28 0.38 0.31 0.21 0.02

0.643 0.566 0.325 0.419 0.639

0.678 0.583 0.423 0.422 0.499

0.541 0.616 0.463 0.454 0.559








0.819 1.79

0.815 1.55

0.826 2.15

0.010 0.76

– –

– –

– –







TSW—thousand seed weight; T50—time (h) to reach 50% of final germination; Total germination—percentage of germinated seeds; Growth rate—an average of daily increase in shoot length (cm/24 h) recorded during following 10 days; Leaves no and Shots no—shoots and leaves number calculated for 4-week old plants; FW—fresh weight (g) and DW—dry weight (g) of leaves from 4-week old seedlings; Roots formation expressed their length (cm) measured for 9-week old plants; Fv /Fm —the maximum quantum yield of PSII and PI—the performance index, both measured before prehardening (Supplementary Fig. A1A); Resistance—the frequency (%) of plants that had regenerated shoots on the 14th day of regrowth after infection.

cultivar-dependent resistance with the specific improvement of photosynthesis efficiency during plant hardening. The changes in the photosynthetic efficiency measured during cold acclimation are often measured by the chlorophyll a fluorescence (Huner et al., 1993; Taulavuori et al., 2000; Rapacz et al., 2004; Ensminger et al., 2006). In our studies, the photosynthesis efficiency was evaluated based on the maximum quantum yield of PSII (Fv /Fm ) and the performance index (PI). The parameters were compared for triticale cv Modus (able to develop partial resistance after cold treatment) and SaKa3006 (susceptible to infection despite seedling hardening) after pre-hardening (12 ◦ C for 7 days) and hardening (4 ◦ C for 14 and 28 days) periods. These two parameters showed different patterns of the changes during cold treatment (Fig. 2A and B). The values of Fv /Fm increased slowly but continuously, with a prolongation of the hardening time (Fig. 2A), whereas PI reached the maximum values already after two weeks of hardening (Fig. 2B). The values of both Fv /Fm and PI were genotype-dependent. The significantly higher values were measured for cultivar Modus than for cultivar SaKa3006 in almost each (pre)hardening time point. Moreover, PI differences between parental genotypes were more considerable in comparison to Fv /Fm . Fig. 2D, E, G, H, J and K (and Supplementary Fig. 3A and B) show the relationships between the chlorophyll a fluorescence parameters and resistance for 95 lines of the DH population. Fv /Fm and PI were measured before (pre)hardening (Supplementary Fig. 3 A and B), after the seedling pre-hardening (Fig. 2D and E), after 2 weeks of hardening (Fig. 2G and H), and after 4 weeks of hardening (Fig. 2J and K), whereas resistance was expressed as a percentage of the regenerated plants that were hardened for 4 weeks, then infected with M. nivale and allowed to regrow in optimal conditions. The values of both parameters of chlorophyll a fluorescence were enhanced gradually with resistance improvement in particular DH lines. Similarly, to the changes for parent seedlings (Fig. 2A and B), the prolongation of hardening time increased the values of Fv /Fm (the averages of the whole population were 0.816, 0.819, 0.826, for Fig. 2D, G and J, respectively), whereas, the maximum of

the PI values was reached already after 2 weeks of hardening (the averages of the whole population were 1.78, 2.37, 2.3, for Fig. 2E, H and K, respectively). Peroxidases activity as a part of the photoprotection mechanisms during hardening Plant peroxidases (PXs) are important components of the antioxidative system, maintaining the balance and uninterrupted functioning of the chloroplast and plant cell by control of the ˛ hydrogen peroxide concentration (Dabrowska et al., 2007). These enzymes can use different electron donors and include the ascorbate peroxidases (APXs) and gluthatione peroxidases (GPXs), and ´ can further be followed by peroxiredoxin (Szechynska-Hebda and ´ Karpinski, 2013). Total peroxidase activity was measured according to the method of hydrogen peroxide scavenging in samples, without distinguishing particular enzyme types (Lück, 1962). The results showed that PX activity was lower during the early prehardening period (7 days at 12 ◦ C, Fig. 2C and F) in comparison to the control from 20 ◦ C (Supplementary Fig. 3C), whereas it increased considerably at the 14th day of hardening period (Fig. 2C and I). Susceptible SaKa3006 and resistant Modus did not differ in PXs activity measured on 14th day of hardening (Fig. 2C). In contrast, further prolongation of the hardening period has increased the PX activity only in the Modus plants. A similar pattern was determined for 95 lines of the DH population. Enzyme activity was enhanced along with cold treatment for the resistant lines, but not for the susceptible ones (Fig. 2I and L). The slope for the trend lines was particularly higher at 28 day of hardening (Fig. 2L) and was similar to those determined for Fv /Fm and PI (Fig. 2J and K, respectively). Adjustment in growth-related traits at post-infection period The positive correlation between cold-induced resistance and photosynthesis efficiency as well as antioxidative enzymes


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Fig. 2. Parameters of chlorophyll a fluorescence (A, D, G, J for Fv /Fm and B, E, H, K for PI) and peroxidase activity (C, F, I, L) (absorbance g−1 protein) of parents SaKa 3006, Modus (A, B, C, respectively) and lines of DH population derived from cross between SaKa 3006 and Modus (D–L). The parental plants were prehardened (preH) and hardened 14 and 28 days (14H and 28H). Similarly treated were DH lines: prehardened (D–F), 14 days hardened (G–I) and 28 days hardened (J–L). Significance levels according to t-Student test: * P ≤ 0.05; ** P ≤ 0.01.

during plant hardening can suggest an enhanced level of the photosynthetic production and the better acclimation to stress of the resistant DH lines. This can positively influence metabolic activity and provide the plant with a faster and more successful regeneration process after infection. Indeed, improvement of

traits related to biomass accumulation (number of leaves, FW and DW) was shown in resistant lines of DH population (Fig. 3, Supplementary Fig. 4). A positive correlation was calculated when the plants were treated with cold but not infected with M. nivale (Supplementary Fig. 4). Similarly, in experiment involving infection

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Fig. 3. Morphological parameters of 95 lines of DH population derived from cross between SaKa 3006 and Modus in post-infection period. Lines were derived from cross between SaKa3006 × Modus. Plants were prehardened and hardened 28 days, then infected with M. nivale and allowed to regrowth 14 days in optimal conditions. Leaves number (A), fresh weight (g) (B) and dry weight (DW, g) (C) were determined.

step, the DH lines regenerating a higher percentage of seedlings simultaneously showed faster regrowth of the survived plants: a higher number of leaves, higher FW and DW per regenerated plant (Fig. 3). The highest values of the leaves number, FW and DW were measured for exactly the same DH lines in both experiments. However, both the slope of trend lines and the regression coefficients were higher for the infected than for uninfected plants (Fig. 3 and Supplementary Fig. 4, respectively), indicating that infection can amplify the differences between susceptible and resistant DH lines in biomass accumulation. Therefore, it can be concluded that effectiveness of photosynthesis in a genotype-dependent manner is a factor that determines not only a higher probability of plant survival after infection (plant number), but also the fitness and regrowth potential of the regenerated seedlings (their regrowth rate). QTL mapping and metaanalysis of PSII- and PXs-related transcripts Resistance of winter crops to Microdochium nivlae is complex and quantitatively inherited; the progress of the infection depends on the genotype and the host genotype × environment interac´ ´ tion (Table 1; Szechynska-Hebda et al., 2010; Szechynska-Hebda et al., 2013). To verify the genetic relation within studied traits, the QTL analysis was performed for 95 lines of the SaKa3006 × Modus population and their parents. For traits related to chlorophyll a fluorescence (Fv /Fm , PI), peroxidase activity, leaves number, fresh and dry weight, the total number of the 99 QTLs were mapped on all chromosomes, except 4B and 1R (Supplementary Tables 1–3). The number of QTLs, significantly associated with plant resistance to M. nivale, was 38 and included the genomic regions on chromosomes: 1A, 2A, 3A, 4A, 6A, 7A, 1B, 2B, 3B, 5B, 6B, 2R, 3R, 4R, 5R, 6R, 7R (Supplementary Tables 1–3). The detailed QTL analysis identified several chromosomal regions that were shared by QTLs involved in the adjustment of photosynthesis during hardening, improvement of traits related to biomass accumulation, and regulation of cold-induced resistance (number of plants regenerated after infection). SMA and CIM analysis revealed main QTLs for photosynthesis-related traits, covering regions on 5B chromosome for Fv /Fm and PI and regions on 7R chromosomes for PI (Table 2, Supplementary Figs. 5A–F, and 7A and B). The region on 5B chromosome, which controls simultaneously Fv /Fm and PI for the pre-hardened, 2- and 4-weeks hardened seedlings was located between markers loci tPt-0228 (26.868 cM) to wPt-7848 (46.110 cM) (Table 2). Additionally, QTL related to Fv/Fm was found on chromosome 7R between markers: wPt-1420 (26.868 cM)–wPt-9872 (26.868 cM), and QTL related

to PI was determined on chromosome 7R at the molecular markers range from rPt-509329 (19.042 cM) to wPt-3379 (23.387 cM) (Table 2). All quantitative trait loci detected for Fv /Fm and PI showed a negative effect, therefore the alleles came from more resistant cultivar Modus (Supplementary Table 1). The QTLs with high LOD scores and R2 (%) for traits associated with plant survival after infection (resistance) were identified on chromosome 5B in the marker interval: wPt-1420 (26.868 cM)–wPt-9872 (26.868 cM) (Table 2, Supplementary Fig. 6, Supplementary Table 1). QTLs for biomass accumulation (number of leaves on regenerated plants, their fresh and dry weight) shared the chromosomal region with QTL for resistance. Moreover, QTL for traits associated with the plant survival after infection was colocated with the QTL linked to Fv /Fm in all hardening periods, but it did not coincide with any locus for PI (Table 2). All identified QTLs had negative allelic effects; a negative effect implied a lower value for the trait conferred by the Modus allele (Supplementary Table 1). The second region associated with the plant resistance was located on chromosome 7R within the marker interval: rPt-509329 (19.042 cM) to wPt-3379 (23.387 cM) (Table 2, Supplementary Fig. 7). Similarly as for chromosome 5B, QTL for regeneration was a part of the chromosome region that covered QTL for the number of leaves per regenerated plants, but in contrast, QTLs for FW and DW were not detected in the same region. This QTL coincided also with the major QTL for PI (Table 2) for plants hardened up to 2 weeks. QTLs identified for resistance and number of leaves on chromosome 7R had positive allelic effects, and a positive effect implied a higher value for the trait conferred by the SaKa3006 allele (Supplementary Table 1). The coincidence between QTLs for peroxidases and chlorophyll a fluorescence were found on chromosome 5B (Table 2, Supplementary Fig. 8). The marker wPt-5514 (55.128 cM) indicated a region of the QTLs for peroxidases (preH and 28H), the Fv /Fm (preH, 14H and 28H) and PI (14H and 28H) (Table 2). For plants hardened 4 weeks, additional QTLs related to peroxidases were identified in the marker intervals from wPt-1420 (26.868 cM) to wPt-9872 (26.868 cM) and from tPt-0228 (26.868 cM) to wPt-7848 (46.110 cM). They were shared with QTLs for Fv /Fm and PI, respectively. Moreover, the QTLs for peroxidases shared the same regions on chromosome 5B with QTLs for traits: number of leaves, FW and DW. On chromosome 7R, the coincidental QTLs for PXs and traits associated with PI, resistance, and number of leaves per regenerated plant were detected between marker intervals: rPt509329 (19.042 cM) to wPt-3379 (23.387 cM) and at region of A40M57 189 marker (45.812 cM). For each QTL the Modus allele served to increase PX activity at 28 days of hardening. In contrast, the SaKa3006 allele served to alternation of the PX activity that


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Table 2 The coincidence between QTLs on chromosome 5B and 7R for DH population derived from cross between SaKa3006 × Modus. Parameters of chlorophyll a fluorescence (Fv /Fm and PI) and peroxidase activity (PXs) were determined for plants prehardened (preH) or hardened for 14 and 28 days (14H, 28H). Plants resistance, leaves number (leaves no), fresh and dry weight (g) (FW and DW, respectively) were analyzed after M. nivale infection. QTLs were accepted with P value lower than 0.05 (P < 0.05) for SMA analysis and LOD higher than 2.5 for CIM analysis. Details of the SMA and CIM analysis are presented in Supplementary table A1 and Supplementary Fig. A5–A8.

PI preH PI 14H PI 28H

tPt-0228 wPt-3569 wPt-8637 wPt-1589 Xbarc004 tPt-513459 wPt-7848

Leaves no

tPt-0228 wPt-3569 wPt-8637 wPt-1589 Xbarc004 tPt-513459 wPt-7848

FW DW PXs preH wPt-5514 PXs 28H

leaves no

PXs 14H

PI 14H

PI preH

A40M57 _189


wPt-1420 wPt-9724 wPt-8604 wPt-9666 tPt-513029 wPt-9872

PXs 28H

PI 28H

PI 14H


PI preH

PXs 28H

Fv/Fm 28H

Fv/Fm 14H


5B Fv/Fm preH



rPt-509329 rPt-401635 rPt-399990 rPt-389980 tPt-513136 Xwmc161 rPt-509033 rPt-507762 rPt-509596 rPt-402501 rPt-508478 rPt-389395 wPt-0749 wPt-3379

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cold acclimation








2,5 -2,5


Pearson's r 0,7 (0.3*) genes expresion (log(2) ratio)






cold acclimation fungal infection








0,05 peroxidases




defence responces Fig. 4. The alteration in the expression of transcripts related to PSII (A) and peroxidases (B) during cold acclimation and resistance induction. The set of the microarray experiments involving 120 perturbations and the total sample number 490 was chosen for metaanalysis; 104 perturbations were selected from experiments focused on the fungal infection, and 16 were chosen from the experiments concerning the cold treatments (details in Supplementary Fig. A.9 and A.10). (A) Calculation of the Pearson coefficient, between an expression of 79 transcripts involved in cold acclimation and the same transcripts during fungal infection, was based on logarithm values obtained according to the Hierarchical Clustering. The Pearson coefficient for 83 studied transcripts is shown in bracket (including 4 of the most scattered points on graph). (B) Average differences in the level of the transcripts related to expression of the total peroxidases (46), ascorbic peroxidases (APX, 12), glutathione peroxidase (GPX, 12) and peroxiredoxins (PRX, 13).

was measured at pre-hardening period and 14 days of hardening (Supplementary Table 1). The mapping of markers and analysis of QTLs provided some evidence, that cold acclimation and cold-induced resistance to fungal pathogens can influence the similar chromosome regions. The most of QTLs that were shared by studied traits were located on the wheat chromosome 5B and the alleles came from more resistant cultivar Modus. To further validate the dependence between cold acclimation and cold-induced resistance as well as the role of photosynthesis and peroxidases, we performed metaanalysis of the microarray experiments using Genevestigator tools. Metaanalysis based on Affymetrix Wheat Genome Array data was conducted for winter wheat for 120 perturbations; 104 were related to fungal infection, and 16 were chosen for the conditions inducing cold acclimation. Using the hierarchical clustering tools, we found, that transcripts related to the photosystem II have a very similar expression profile, when cold treatment was compared to plant infection with fungal pathogens (Supplementary Fig. 9). More of the PSII-related transcripts were down-regulated during both stresses. The positive linear correlation for particular transcripts induced either by cold treatment or by infection was obtained, and the Pearson’s correlation coefficient was 0.7 and 0.3 for 77 and 83 transcripts, respectively (Fig. 4A). Four transcripts differed significantly when cold and infection were compared (Fig. 4A, Supplementary Fig. 9, transcripts indicated with asterisks) and they lowered the value of Pearson’s correlation coefficient significantly. These transcripts were related to the predicted proteins: sterol methyl oxidase, photosystem II light harvesting complex protein, photosystem II subunit R, chlorophyll a-b binding protein 7 ( The same experimental perturbations were applied to verify whether transcripts related to peroxidases were similarly modulated during cold acclimation and defense responses (Fig. 4B, Supplementary Fig. 10). Simultaneous analysis of the multiple microarray experiments showed a set of the transcripts (46), with the specific expression. The most of them were up-regulated by both, cold and infection stress. However, the infection had influenced on the peroxidase transcripts more effectively. Similar results were influenced for the transcripts related to ascorbic peroxidases (APXs) and glutathione peroxidases (GPXs), however, GPXs seems to play a crucial role during cold treatment. In contrast, peroxiredoxins (Prx) transcripts differed between the cold treatment and infection (Fig. 4B).

Discussion The winter survival of a plant is regarded as a complex array of the interrelated events, both with regard to the environment and plant adjustment to stress. Temperature, the snow and ice cover as well as physiological drought and disease are limiting factors, therefore, winter crops have developed various molecular mechanisms of acclimation. Here, we showed that the winter plant, at least triticale, is able to integrate the cold acclimation and defense pathways against winter pathogens via the regulation of the photosynthesis-related features in a physiological and genetic manner. Two cultivars of hexaploid winter triticale (×Triticosecale), namely Modus and SaKa3006, have been previously characterized with respect to their differing cold-induced responses to infection with the fungal pathogen M. nivale. Modus was able to develop resistance after hardening, whereas SaKa3006 plants were susceptible to fungal infection despite cold treatment (Supplementary ´ Fig. 2; Szechynska-Hebda et al., 2011). Significantly different efficiency of the cold-induced resistance of the parental genotypes allowed to generate a mapping population consisting of 95 doubled haploid lines from a cross between SaKa3006 × Modus. In case of traits related to cold-induced resistance, transgressive segregation among the DH lines was apparent (Table 1). One of the most significant factors that predetermined the plant successful survival during infection was the rate of growth and biomass accumulation of the germinated seedlings (Table 1; Enright and Cipollini, 2007). The time required to reach 50% germination was shorter approximately 14 h, and the daily shoot growth rate and dry weight of 4-week-old seedlings were 30% higher for the resistant parental cultivar (Modus) than for the sensitive cultivar (SaKa3006). Other traits related to the formation of shoots, leaves and root were also improved about 13–22% for resistant genotypes. Similarly, significant positive correlations between resistance and growth-related traits were found for 95 lines of the DH population (R2 > 0.50). Fast germination (Table 1) and early achievement of physiological maturity (Fig. 1) resulted in two capabilities of the resistant seedlings. First, physiological and metabolic maturity of the chloroplasts and earlier developing leaves (Fig. 1) enabled the plants to engage in effective photosynthesis much faster and generate a supply of the energy for production of the metabolites involved in plant defense. Indeed, chlorophyll a fluorescence


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parameters were correlated relatively strongly with plant germination, biomass accumulation and seedling resistance (Table 1). Secondly, if leaves development can quickly progressed, the reinforcement of the leaf also is completed earlier. This factor is particularly important for both, the cold acclimation (Ruelland et al., 2009) and basic-type defense responses (Amid et al., 2012; ´ et al., 2013). In resisYeats and Rose, 2013; Szechynska-Hebda tant cultivar, the fast synthesis of lipid-like substances (fats, waxes, cutins, alkaloids, terpenes, glycosides and phenols) improved the epidermis surface in submicron- and micron-scale roughness, and resulted in the leaf superhydrophobicity and restriction of fungal adhesion. Additionally, lignin was shown to strengthen the ´ cell wall in internal tissues of the leaf (Szechynska-Hebda et al., 2013). Hardening also increased the cellulose/hemicellulose ratio in leaf tissues of resistant plant. Cell wall structure rich in cellulose, is dense, well-ordered and chemically-resistant, and can more effectively protects leaves against diffusion of the fungal ´ enzyme and tissue digestion (Szechynska-Hebda et al., 2013). The specific physical and chemical properties of the plant tissue can influence the strategy of fungal invasion (Ellis et al., 2002; Gaskin et al., 2005; Bhushan and Jung, 2008; Teisala et al., 2011). Indeed, M. nivale was shown to be able to invade triticale by biotrophic interactions with the resistant plants (fortified cell wall), whereas by necrotrophic interactions with ´ et al., susceptible tissue (unfortified cell wall) (Szechynska-Hebda 2013). Dependence of the plant growth and biomass accumulation on photosynthesis is obvious (Table 1; Evans and von Caemmerer, 2011). Photosynthesis changes during cold acclimation and the acclimation-related processes that can function efficiently only in the presence of light were presented (Ruelland et al., 2009; Yamori et al., 2009, 2010, 2014; Janda et al., 2014). Furthermore, correlation between resistance to diseases and photosynthesis was shown and discussed (Table 1; Evans, 2013; Kangasjärvi et al., 2012). Nevertheless, the cold-induced adjustment of the photosynthesis processes that triggers the defense mechanisms against fungal pathogens was not studied in details. Here, we showed a high positive correlation between the photosynthesis efficiency during cold hardening and cold-induced resistance. Chlorophyll a fluorescence parameters were higher for cultivar Modus, which was able to develop resistance after cold treatment and similarly, these universal markers increased almost linearly with the improving resistance of the particular DH lines (Fig. 2). The differences between Modus and SaKa3006 and the slope of the trend lines for the DH lines, were the highest after 28 days of hardening (Fig. 1A, B, J and K). It was shown earlier, that 28 days of hardening can result in a fully developed defense mechanism only in the coldresponsive cultivars, and then, such period can distinguish resistant ´ genotypes from susceptible ones (Szechynska-Hebda et al., 2013). Presently, this result, at least in part, can be explained by the role of photosynthesis in resistance development during cold conditions. The proper photosynthesis can only be maintained through the activity of a signaling network that responds to the actual status of the cell. However, the modulation of cellular metabolism, transcriptome and proteome during cold acclimation is very complex and in many cases highly specific (reviewed by Ruelland et al., 2009). Despite this, it is known that cross-talk between different abiotic and biotic stresses often occurs. Therefore, in our studies, we chose universal stress factors describing the metabolic pathways that can be common for both, acquired acclimation to cold and cold-induced resistance to pathogens. One of these, Fv /Fm , is a sensitive indicator of the competence of PSII photochemistry and photosynthetic electron transport (PET) (Baker, 2008; Rosso et al., 2009), and thus it reflects different stress conditions. Low temperatures in light conditions increase

the excitation pressure in PSII (Ruelland et al., 2009), but reduce the rate of PET (measured as Fv /Fm , Fig. 2A), and consequently, can induce overproduction of reactive oxygen species (ROS), and lead to destruction of the photosynthetic apparatus and photoinhibition (Rizza et al., 2001; Takahashi and Murata, 2008; Tyystjarvi, 2008). In our studies, variation in Fv /Fm mimics a slight (lower values after pre-hardening and 14 days of hardening), but reversible (reversed at 28 day of hardening) photoinhibitory effect during cold treatment (Fig. 2A). This acclimation of the photosynthetic apparatus is a key factor, since, the winter plant species must maintain the capacity for active photosynthesis (Hurry and Huner, 1991; Rizza et al., 2001) and growth (Majláth et al., 2012) during prolonged exposure to low, non-freezing temperatures. The increased tolerance to photoinhibition as a consequence of cold acclimation is not caused by an increased capacity to repair damaged PSII reaction centers or increased non-photochemical quenching. It is rather a result of the increased capacity to keep the quinone QA oxidized (i.e. high photochemical quenching) and the improved rate of PET (Fig. 2, Ruelland et al., 2009; Janda et al., 2014); the result of induction of the genes encoding photosynthetic proteins (Fig. 4A, Rizza et al., 2001; Dal Bosco et al., 2003; Yamori et al., 2014) and enzymes of the scavenger systems for ROS e.g. peroxidases (Fig. 4B, Huner et al., 1998). Moreover, the data presented here showed, that also total activity of the peroxidases, involving at least APXs, PRXs and ´ and GPXs in the chloroplasts and cytoplasm (Szechynska-Hebda ´ Karpinski, 2013) mirrored the Fv /Fm changes (Fig. 2). In the control plants, kept at 20 ◦ C, the peroxidase activity of the resistant plants was similar or lower in comparison to the susceptible plants (Supplementary Fig. 3C), however, the activity determined during cold hardening increased considerably for the resistant cultivar Modus and the DH lines (particularly after 28 days of cold hardening, Fig. 2L). A different pattern of changes for the second universal parameter of chlorophyll a fluorescence, i.e. the performance index, results from its function as a comprehensive indicator of various components influencing photosynthetic efficiency. The PI has three components. The first component shows the force due to a concentration of active reaction centers. The second component is the force of the light reactions, which is related to the quantum yield of primary photochemistry. The third component is the force related to the dark reactions (Kalaji and Guo, 2008). The integration of different components makes the PI useful in studies of longterm plant responses to cold, as cold acclimation is much more complex than changes in or around PSII. For example, together with PSII, also PSI activity was shown to decline after a low light cold treatment. Moreover, the increased capacity to keep QA oxidized appears to be a consequence of cold-induced stimulation of mRNA and protein levels associated with the major regulatory enzymes of photosynthetic carbon metabolism (Ruelland et al., 2009), or alternatively, a result of shifting protein expression to produce isoforms with improved performance at low temperature (Yamori et al., 2014). Our results showed that in contrast to Fv /Fm , PI increased during 14 days of hardening when compared to plants untreated with cold (Fig. 2, Supplementary Fig. 3B) and was stabilized after such period of time (Fig. 2). These data and results of significantly higher PI values for the resistant DH lines (Fig. 1E, H and K) suggest a better balance (stabilization) of the photosynthetic light-to-dark reaction during (pre)hardening. Thus, winter triticale appears to adjust the number of electron consuming sinks in response to low temperatures. The efficient balancing of the light/dark reaction can maintain the plant vitality also during infection in cold conditions, and furthermore determine plant regrowth and productivity at the post-disease period. In fact, the resistant DH lines, with the improved Fv /Fm and PI during hardening, had a higher number of leaves as well as enhanced FW and DW per regenerated plant during the regrowth period after both,

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hardening alone (Supplementary Fig. 4), and combined stresses of the cold and infection (Fig. 3). However, the combined abiotic and biotic stresses have amplified the differences between susceptible and resistant plants in the post-disease biomass accumulation in comparison to the cold treatment only. Contact with pathogen often leads to the development of chlorotic and necrotic areas. The quick regrowth causes an increased demand for assimilates in the plant, and is an additional energy-consuming factor. Therefore, the partitioning of assimilates and source–sink regulation must be under tight control during the combined stress. In our experiments, the plants treated with the cold only and plants that were hardened and then infected were all cut after stress treatments. The new leaves had developed from the crown during recovery period. Thus, it suggests that the potential to induce the faster regrowth of the resistant plants was maintained in the crowns (the source of energy, when leaves are damaged), however such mechanisms required further detailed studies. In addition to the traditional role of photosynthesis in energy transduction, the redox state of the photosynthetic apparatus might also act as a sensor of actual environmental conditions that trigger the retrograde signals from the chloroplasts to the ´ and nucleus (Pfannschmidt et al., 2001, 2009; Szechynska-Hebda ´ ´ Karpinski, 2013; Karpinski et al., 2013). More rapid or stronger accumulation of the photosynthesis-originated signal molecules, i.e. H2 O2 and sugars in the resistant plant (Yang et al., 2013; Thakur and Sohal, 2013) was shown to regulate the defense´ related genes (Szechynska-Hebda et al., 2010; Xiao et al., 2000; ´ ´ and Karpinski, 2013). Thus, the plants can adjust Szechynska-Hebda different signals controlling nuclear genes in order to establish a photosynthesis-dependent cold-acclimated state (Kurepin et al., 2013), that influence further responses, e.g. to biotic stresses. Our studies for the SaKa3006 × Modus mapping populations, indicate that certain groups of genes controlling the mechanisms of acclimation to cold, resistance to fungal pathogen and biomass production were coincidentally regulated by the photosynthesisinduced signals. The chromosomes 5B and 7R were found to contain QTLs shared the same positions within traits related to: changes in the parameters of chlorophyll a fluorescence during cold, cold-dependent leaves development, FW and DW accumulation, cold-regulated peroxidase activity and cold-induced resistance against the fungal pathogen M. nivale (Table 2). For most of the QTLs, the resistant Modus allele served to increase the traits’ positive alternations. However, the QTLs for chlorophyll a fluorescence parameters, that describe different physiological events (Fv /Fm and PI), were a part of QTLs related to a plant resistance at different locations. It suggests that balancing of the PET in cold-inducted resistance can be controlled independently from the integration of light reactions with dark reactions. Genetic analyses of chlorophyll a fluorescence and chlorophyll content were, up to this time, conducted on wheat, and the most of the previously referenced QTL analyses were performed at late growth stages (Liang et al., 2010; Zhang et al., 2009). Similarly, QTLs related to an increase of the FHB resistance were reported at different wheat chromosomes: 2A (Garvin et al., 2009), 3A (Chen et al., 2007), 7A (Kumar et al., 2007), 6B, 7B, 4B (Buerstmayr et al., 2012). However, according to our knowledge, we showed for the first time, that cold integrates at genetic level: (1) the photosynthesis efficiency, followed by the biomass accumulation; (2) mechanisms of the cold acclimation and cell/chloroplast photoprotection, at least by ROS balancing by peroxidases; and (3) responses restricting disease. Since, the coincidence of QTLs for various traits, with allelic differences corresponding to the expected relationship between the traits, constitutes a strong evidence that these traits are causally related (Thumma et al., 2001; Yin et al., 2010), we suggest, that balancing of photoinhibition/photoprotection


responses is a key factor in cold-induced resistance to the winter pathogen. This statement was also confirmed by a simultaneous analysis of the multiple microarray experiments. The results showed a set of common genes that are similarly induced or suppressed during both processes, cold acclimation and cold-induced resistance. The expression pattern of genes related to the photosystem II, was very similar to the changes of particular genes during infection with fungal pathogens (Fig. 4A, Supplementary Fig. 9). This indicates that similar genes could play an important role in PSII adjustment to abiotic and biotic stresses. In the same way, the regulation of major system detoxifying the hydrogen peroxide in plant cells i.e. the ascorbate-glutathione cycle, can be a common mechanism during cold and infection (Fig. 4B, Supplementary Fig. 10). The ascorbate peroxidase (APX), glutathione peroxidases (GPX), and peroxiredoxins (PRX) have eliminate the chloroplast-originated H2 O2 , however, their function is more complex, since cold treatment up-regulated the GPXs transcripts more efficiently, whereas the infection with fungal pathogens induced mainly, APX and PRX. In conclusion, low temperatures and light conditions can induce the genotype-dependent resistance mechanisms against the fungal pathogen M. nivale. The genotypes able to develop effective defense mechanisms are flexible in their photosynthesis process. The physiological and genetic crosstalk between cold acclimation and cold-induced resistance involve the adjustment of the reactions in the PSII proximity and a more efficient integration of the primary photochemistry of light reactions with the dark reactions; followed by an induction of photoprotective mechanisms (at least, peroxidases). These mechanisms allow resistant plants improve growth and development, and as a consequence, avoid the developmental stages that are sensitive to fungal infection and regrow quickly at the post disease period. Although, photosynthesis-related processes play a key role in the enhancement of cold acclimation and cold induced resistance; the light at low temperature induces several processes, which are not directly related to photosynthetic adjustment and/or cold acclimation mechanisms. Recent results showed that light, and light in combination with cold may affect the changes in e.g. certain plant hormones (auxin, cytokinin, SA, ethylene), stress-related protective substances (proline, phenolics, polyamine) and antioxidative enzymes other than peroxidases, as well as the expression of stress-related genes and various kinds of transcription factors (reviewed in detail by Janda et al., 2014). It is also known, that high light can induce plant resistance in ´ temperature optimal for growth (Szechynska-Hebda et al., 2010). Therefore, this complexity of plant’s responses indicates either the cross talk between chloroplast-originated signaling and other protective mechanisms, or different independent pathways that are responsible for cold-induced resistance; and it must be considered in further studies. Contributions and acknowledgements This work was supported by the project NCN N N310 778640. MW received grant support. MSH and MW designed the experiment. MSH and IW analyzed the results, performed QTL analysis and prepared the manuscript. MW reviewed and approved the final manuscript. MSH, GG, ED, IZ˙ analyzed morphological traits. MSH performed microscopic analysis, photographic documentation and the GV metaanalysis. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at 2014.12.017.


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Photosynthesis-dependent physiological and genetic crosstalk between cold acclimation and cold-induced resistance to fungal pathogens in triticale (Triticosecale Wittm.).

The breeding for resistance against fungal pathogens in winter triticale (Triticosecale Wittm.) continues to be hindered by a complexity of the resist...
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